Many bioanalytical methods are based on the oxidative status of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). NAD has a multiple ringed structure, which undergoes redox reactions within its nicotinamide ring. The closely related NADP molecule is phosphorylated on the 2′ position of the adenosine ribose ring.
NAD and NADP can be reversibly reduced by the formal addition of hydride ions and both molecules act as coenzymes in reversible reactions. Accordingly, enzymatic reactions based on NAD and NADH are amenable to fluorescent analysis.
Many oxidoreductase enzymes can use these cofactors to transfer hydrogen groups between molecules. Because the reduced forms of these molecules differ from their oxidized forms in their ability to absorb light, reactions have been quantitated based on light absorption at 340 nm or by fluorescent emission of light at 445 nm.
Enzymatic dehydrogenase reactions can take advantage of the property of the reduced forms of NAD and NADP to absorb light at a wavelength of 340 nm while the oxidized form does not. Similarly, the reduced forms are capable of fluorescent emission at 445 nm when excited at 340 nm, while the oxidized forms are not. These properties permit quantitation of reactions that directly involve a change in the oxidative state of these cofactors. For example, when phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase are used to catalyze the formation of NAD from NADH in the presence of adenosine triphospate (ATP), the concentration of adenosine triphosphate can be measured as a decrease in fluorescence intensity (U.S. Pat. Nos. 4,446,231 and 4,735,897).
Oxidoreductases are also quite popular in the quantitative measurement of blood glucose levels, see for example EP 0 293 732A2, US 2005/0214891 A1, and US 2006/0003397. All of these publications describe a similar test protocol for measurement of glucose, in which a reagent system containing the enzyme-coenzyme pair glucose dehydrogenase (GlucDH)/nicotinamide adenine dinucleotide (NAD) is used. Upon action of GlucDH, a hydride ion is transferred from glucose to NAD, such that NADH forms. The resulting quantity of NADH is directly correlated to the concentration of glucose. NADH is a strong fluorophore, whose concentration can be determined by a measurement of the fluorescence intensity. Analyte concentration in a sample is typically determined by correlating the fluorescence intensity measured to a calibration curve obtained with known analyte concentrations.
Evidently, enzyme-based measuring systems for biochemical analytics are important components of clinically relevant analytical methods. This primarily concerns the measurement of analytes e.g. metabolites or substrates which are determined directly or indirectly with the aid of an enzyme. The analytes are converted with the aid of an enzyme-coenzyme complex and subsequently quantified. In this process the analyte to be determined is brought into contact with a suitable enzyme and a coenzyme where the enzyme is usually used in catalytic amounts. The coenzyme is changed e.g. oxidized or reduced by the enzymatic reaction. This process can e.g. be detected photo metrically. A calibration provides a direct correlation between the measured value and the concentration of the analyte to be determined.
Coenzymes are organic molecules which are covalently or non-covalently bound to an enzyme and are changed by the conversion of the analyte. Prominent examples of coenzymes are nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) from which NADH and NADPH respectively are formed by reduction.
Many oxidoreductase-based measuring systems known from the prior art have a limited shelf-life and require cautious handling such as cooling or dry storage in order to achieve sufficient storage life. Erroneous results caused by incorrect, unnoticed, faulty storage can therefore occur. In particular the exhaustion of desiccants due to opening of the primary packaging and long-time use periods can result in measuring errors.
Both the essential components of such enzyme-based measurement systems, i.e. the enzyme the coenzyme can independently contribute to such limited stability. For example coenzymes such as NAD and NADP are known to be rather unstable.
Both NAD and NADP are base-labile molecules the degradation paths of which are described in the literature (see e.g. N. J. Oppenheimer in The Pyridine Nucleotide Coenzymes Academic Press, New York, London 1982, J. Everese, B. Anderson, K. Yon, Editors, chapter 3, pages 56-65). Essentially ADP-ribose is formed during the degradation of NAD or NADP by cleavage of the glycosyl bonds between the ribose and the pyridine unit. The reduced forms NADH and NADPH are acid labile; e.g. epimerization is a known degradation path.
The instability of NAD/NADP and of NADH/NADPH is due to the lability of the glycosyl bond between the ribose and the pyridine unit. But even under conditions that are not drastic such as in aqueous solution, the coenzymes NAD and NADP may already be hydrolysed solely due to ambient humidity.
CarbaNAD is an analogue to NAD, wherein ribose is substituted by a carbacyclic sugar unit. CarbaNAD (or Carba-NAD) has the following structure (I):
However, even when using the more stable coenzyme carbaNAD, an array of quite fundamental problems is connected to the measurement of fluorescence intensity including the following:
One important source of errors for measurements based on fluorescence intensity measurements comes from nonspecific light, which reaches the detector from the environment and can cause an unspecific signal.
The intensity of the measured fluorescence light is not only a function of the quantity of the fluorophore. Rather it is also significantly influenced by its molecular environment in the sample. In particular processes which are summarized under the term fluorescence quenching contribute errors in measurements.
The position and orientation of the molecule can change between absorption and emission because in the statistical mean a time in the order of nanoseconds passes between the excitation of a molecule and the emission of a light quantum. Interfering influences result therefrom in regard to the fluorescence intensity, in particular temperature dependence.
Fluorescence is generally excited by ultraviolet light. Photochemical reactions of the electronically excited state may cause bleaching of the fluorophore. This is a further error source.
On this basis, it is an object of the present invention to propose a method which allows for an improved measurement in particular in regard to the described stability issues, measurement errors and interferences.